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Multiplexed Detection of Sepsis Markers in Whole Blood Using medRxiv preprint doi: https://doi.org/10.1101/2020.11.03.20224683; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Multiplexed Detection of Sepsis Markers in Whole Blood using Nanocomposite Coated Electrochemical Sensors Uroš Zupančič1,2, Pawan Jolly1, Pedro Estrela2, Despina Moschou2, and Donald E. Ingber1,3,4,* 1Wyss Institute for Biologically Inspired Engineering, Harvard University, USA, 2Centre for Biosensors, Bioelectronics and Biodevices (C3Bio) and Department of Electronic & Electrical Engineering, University of Bath, UK, 3Vascular Biology Program and Department of Surgery, Boston Children's Hospital and Harvard Medical School, USA 4Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, USA *Address all correspondence to: Donald E. Ingber, MD, PhD, Wyss Institute at Harvard University, CLSB5, 3 Blackfan Circle, Boston MA 02115 (ph: 617-432-7044, fax: 617-432- 7828; email: [email protected] NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 1 medRxiv preprint doi: https://doi.org/10.1101/2020.11.03.20224683; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. ABSTRACT Sepsis is a leading cause of mortality worldwide that is difficult to diagnose and manage because this requires simultaneous analysis of multiple biomarkers. Electrochemical detection methods could potentially provide a way to accurately quantify multiple sepsis biomarkers in a multiplexed manner as they have very low limits of detection and require minimal sensor instrumentation; however, affinity-based electrochemical sensors are usually hampered by biological fouling. Here we describe development of an electrochemical detection platform that enables detection of multiple sepsis biomarkers simultaneously by incorporating a recently developed nanocomposite coating composed of crosslinked bovine serum albumin containing a network of reduced graphene oxide nanoparticles that prevents biofouling. Using nanocomposite coated planar gold electrodes, we constructed a procalcitonin sensor and demonstrated sensitive PCT detection in undiluted serum and clinical samples, as well as excellent correlation with a conventional ELISA (adjusted r2 = 0.95). Sensors for two additional sepsis biomarkers — C- reactive protein and pathogen-associated molecular patterns — were developed on the same multiplexed platform and tested in whole blood. Due to the excellent antifouling properties of the nanocomposite coating, all three sensors exhibited specific responses within the clinically significant range without any cross-reactivity in the same channel with low sample volume. This platform enables sensitive simultaneous electrochemical detection of multiple analytes in human whole blood, which can be expanded further to any target analyte with an appropriate antibody pair or capturing probe, and thus, may offer a potentially valuable tool for development of clinical point-of-care diagnostics. 2 medRxiv preprint doi: https://doi.org/10.1101/2020.11.03.20224683; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. GRAPHICAL ABSTRACT KEYWORDS: Anti-fouling, electrochemical biosensor, multiplexing, point-of-care diagnostics, sepsis Specific and sensitive quantification of multiple biomarkers for point-of-care (POC) diagnostics applications using whole blood samples has been a holy grail in clinical diagnostics for decades. The greatest need for such devices is the diagnosis of life-threatening medical conditions, such as sepsis, where every hour of delay in administration of the correct antibiotic increases the mortality rate by over 7%.1 Unfortunately, no single sepsis biomarker exhibits appropriate specificity and sensitivity for disease diagnosis, and thus multiplexed detection of biomarker panels has emerged as the preferred POC diagnostic option.2 Among the variety of biosensing platforms, electrochemical (EC) biosensors would be preferred to use for this application due to their potential for optimal detection limits, cost-effective integration with diagnostic readers, and low power instrumentation requirements.3, 4 However, while individual biomarker detection has been demonstrated in whole blood using electrochemical systems,5, 6 it only has been possible to carry out multiplexed detection by prefiltering the blood, capturing and then releasing the biomarker molecules,7 which adds greatly to the complexity of a potential 3 medRxiv preprint doi: https://doi.org/10.1101/2020.11.03.20224683; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. integrated ‘sample in-answer out’ device. Commercial EC POC platforms have been developed that utilize separate cartridges for different biomarkers (e.g., Abbott’s i-STAT system), but they do not carry out multiplexed protein detection and quantification cartridge.8 Therefore, there is a need for a low-cost and straightforward multiplexed EC platform that can rapidly quantify multiple biomarkers in a complex sample, such as serum, plasma, or whole blood. One of the reasons behind the limited commercial success of affinity-based EC sensors in complex biological samples is the high susceptibility of the EC sensing elements to biological fouling.9 Nonspecific binding can drastically impact resistive and capacitive properties of the sensing element, hampering electrochemical signal transduction.10 We recently demonstrated that this issue can be addressed by coating electrodes with a three-dimensional (3D) nanocomposite containing crosslinked bovine serum albumin (BSA) doped with conducting nanomaterials, such as gold nanowires (AuNWs) or carbon nanotubes (CNTs), and using 3,3′,5,5′-tetramethylbenzidine (TMB) that precipitates locally on the surface of the electrode to quantify binding.11, 12 This matrix allowed excellent electrochemical signal transduction between the gold electrode and the nanocomposite surface, which could be functionalized with antibodies while maintaining conductive and antifouling surface properties.11, 12 Although the nanocomposite demonstrated excellent antifouling characteristics by maintaining its properties after one-month exposure to human plasma, the addition of expensive AuNWs to the matrix can pose a significant challenge towards commercialization of this technology. Thus, in the present study, we replaced AuNWs with reduced graphene oxide nanoflakes (rGOx) with known antifouling and electrochemical attributes, which are also much less expensive.13, 14 By doing so, the cost of the nanocomposite was reduced by ~99%, which should enable this technology to have an immediate commercial impact. The EC sensor platform we present here, which is 4 medRxiv preprint doi: https://doi.org/10.1101/2020.11.03.20224683; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. enabled by the graphene oxide nanoflakes-based antifouling nanocomposite, can quantify multiple biomarkers and can be easily incorporated with microfluidics. An antibody-based sensor for one of the most important sepsis biomarkers, procalcitonin (PCT), is characterized in serum and whole blood, and the sensor is validated using clinical samples. The broad applicability of the nanocomposite is further demonstrated by the incorporation of two additional sensors for two other infection biomarkers that are not based on antibodies as capture probes: C-reactive protein (CRP),15 which is based on use of a phosphorylcholine (PC) capturing probe, and pathogen- associated molecular patterns (PAMPs) that are bound by immobilized Fc-Mannose Binding Lectin (Fc-MBL).16 This is the first time a multiplexed EC sensor for these three different sepsis biomarkers has been demonstrated in whole blood. RESULTS AND DISCUSSION Nanocomposite characterization Nanocomposite coatings composed of BSA and AuNWs cross-linked with glutaraldehyde (GA) have been shown to demonstrate excellent antifouling properties, maintaining high conductivity even after one-month exposure to human plasma and allowing sensitive detection of interleukin-6 (IL-6).17 To lower the cost of the nanocomposite, AuNW were replaced with conducting rGOx nanoflakes that were terminated with either a carboxyl or amine group. We first constructed an EC sensor using this modified nanocomposite coating and analyzed its ability to detect IL-6 in undiluted serum to compare its sensitivity relative to the AuNW sensor studied in the past and confirm its functionality. The EC sensor employing the nanocomposite with carboxyl
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